Myosin is a family of mechanochemical proteins that demonstrate force- generating ATPase activity when they interact with actin filaments. Although best studied in muscle contraction, conventional myosin (myosin II) in nonmuscle cells appears to be involved in diverse cellular processes including cytokinesis, capping of surface receptors and chemotaxis. Individual myosin heavy chain (MHC) isoforms are encoded by different genes and their expression is tightly associated with cell growth and differentiation. This laboratory has isolated two different cDNAs for nonmuscle MHC, NMHC-A and NMHC-B which are encoded by two different genes in both humans and chickens. An ongoing effort is being made to understand the transcriptional regulation of NMHC-A gene including identification of cis-acting element(s). Previous work has shown that two fragments in the first intron of the human NMHC-A gene, 0.5 kb and 2.8 kb in size, show a 3-10-fold increase in transcriptional activity in NIH 3T3 fibroblasts and C2 myoblasts, but not in differentiated myotubes (using the luciferase reporter system). In addition, it is known that NIH 3T3 cells, myoblasts (C2C12) and glioblastoma (U-138) cells express nonmuscle myosin A relatively abundantly in contrast to C2C12 myotubes and neuroblastoma (SK-N-SH) cells, where the expression is minimal. The 2.8 kb fragment has been progressively narrowed down to a 100 base pair fragment with the most enhancing activity. These fragments were generated using PCR and Luciferase was used as the reporting system. Transfection was carried out using calcium phosphate co-precipitation and the following pattern has emerged: maximal enhancement is seen in NIH 3T3 cells and intermediate activity in myoblasts (C2C12) and glioblastoma (U-138) cells. Minimal activity was seen in neuroblastoma (SK-N-SH) cells. In order to localize the protein binding site, gel shift assays were performed and show specific protein DNA interaction using NIH 3T3 and neuroblastoma nuclear extracts. Sequence comparisons (using GCG) show putative binding sites for SP-1, AP-1 and CTF transcriptional factors (TF) within the 100 bp fragment. However, no competition was seen using the consensus oligos for these TF's, suggesting, therefore, that the protein binding site is distinct. To identify the precise binding site, the 100 bp fragment has been subdivided into overlapping 30 base pair fragments. Methylation interference and mutation experiments are planned.